The long term goal of this study is to understand the molecular sites and mechanisms through which alcohol acts to modulate the function of NMDA receptors. Although NMDA receptors are major CNS targets of alcohol action, the molecular sites and mechanisms of action of alcohol on NMDA receptors are still incompletely understood. Alcohol inhibits NMDA receptors by influencing ion channel gating (1;2), and results of recent studies from this and other laboratories point to sites in the membrane-associated (M) domains in the actions of alcohol. Work from this laboratory has identified and characterized amino acid positions in the third and fourth membrane-associated (M) domains of the NMDA receptor NR2A subunit that influence both gating and alcohol sensitivity of the ion channel; at least two of these positions are putative sites of alcohol action. Although the NR2A subunit predominates in the mammalian brain, recent evidence supports the importance of the NR2B subunit in CNS function (3-6) and alcohol action (7-11). In these studies we will use whole-cell, single-channel, and macropatch concentration-jump patch-clamp recording coupled with mutant cycle analysis and kinetic modeling, as well as molecular modeling and molecular dynamics simulations, to test the central hypothesis that alcohol interacts with specific sites in the M3 and M4 domains to modulate NMDA receptor activity via changes in ion channel gating, and that these sites, and the specific mechanism of inhibition, differ among NR2 subunits and different alcohols. These studies will provide the most complete information to date on the mechanism of alcohol action on the NMDA receptor; specifically, they will provide critical insights into both interaction of ethanol and other alcohols with their sites of action on the NMDA receptor, and the influence of ethanol on NMDA receptor kinetics. By identifying sites in the NMDA receptor gating regions that modulate sensitivity to inhibition by alcohol, as well as sites that are likely to directly bind alcohol, these studies will also identify novel therapeutic targets for the treatment of alcohol use disorders. The knowledge gained from these studies could thus provide a basis for a better understanding of the precise role of the NMDA receptor in the neurophysiological and behavioral effects of alcohol, as well as better pharmacotherapy of alcohol abuse and alcoholism.